Abstract

Fluorogen activating proteins that activate the fluorescence of triarylmethane dyes have been demonstrated as practical tags for both cell surface and intracellular labelling, with applications ranging from single-molecule imaging to whole-animal optogenetics. The binding of a fluorogenic dye can result in thousands-fold activation, serving as a binding mediated optical switch, which activates fluorescence from otherwise dark molecules. Synthesis of various fluorescent donors linked to a far-red excitable fluorogen at distances far shorter than the Forster radius of the dyes has established a new family of FRET -based multi excitation fluorogenic dyes, with tunable excitation and emission properties suitable for use with a wide variety of conventional and superresolution microscopy methods. Use of environmentally sensitive donor dyes produces targeted and activated ratiometric fluorescent indicators, enabling optical physiology at and beyond the diffraction limit. I will discuss applications of pH sensors in living cells for measurement of endolysosomal trafficking and development and validation ROS generating and sensor dyes for use in live cells and model organisms.